Bottom Line:
Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development.Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song.Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.

ABSTRACTThe gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.

pbio-0050321-g004: Variability of Syllable Production in FoxP2 Knockdown PupilsEach vertical column shows the sonograms of five different renditions of the same syllable (scale bar indicates 100 ms, frequency range = 0–8,600 Hz). Each syllable, labeled by a roman numeral, was selected from a different bird. Of note, the first three syllables in (A) (syllables I, II, and III) were imitated from the same tutor as the corresponding syllables in (B) (syllables I′, II′, and III′).(A) FoxP2 knockdown pupils. Two vertical lines mark the beginning and the end of the longest rendition of each syllable to visualize variability of syllable duration (particularly evident in syllables II and IV). Also note the variability in acoustic structure between different renditions of the same syllable (e.g., FM of syllable I, shape and frequency of first element of syllable III, and PG of last element of syllable IV).(B) Syllable duration is relatively invariant in control pupils, as indicated by the vertical lines marking the beginning and the end of each syllable. Acoustic structure is also stable across syllable renditions.(C) Acoustic variability of syllables from rendition to rendition was higher in shFoxP2-injected than in control pupils (shGFP and shControl injections), as indicated by significantly lower syllable identify scores (two-tailed Mann-Whitney U test, *p < 0.05; Bonferroni-corrected α-level). Control and tutor birds sang with comparable variability (two-tailed Mann-Whitney U test, p > 0.8; tutors n = 6, on average 5 syllables per animal; shControl n = 7 animals, on average 4 syllables per animal; shGFP n = 3, on average 5 syllables per animal).(D) Syllable duration varied more from rendition to rendition in knockdown pupils (shFoxP2) than in controls (shControl and shGFP) and tutors, as indicated by a higher mean coefficient of variation of syllable duration (±SEM, two-tailed Mann-Whitney U test, **p < 0.001; Bonferroni-corrected α-level; no difference between tutors, shControl-injected, and shGFP-injected animals, p > 0.7, same animals as [C]).

Mentions:
The comparison of sonograms from different renditions of the same syllable revealed that knockdown pupils sang their syllables in a more variable fashion than control pupils (Figure 4A and 4B). Both the spectral (syllables I and III) and the temporal domain (syllables II and IV) were affected. Of note, the first three syllable examples shown in Figure 4A and 4B (syllables I, II, and III and I′, II′, and III′), stem from different animals, but were learned from the same tutor. To quantify the acoustic variability of syllables, we used the syllable identity score mentioned above. Pairwise comparison between different renditions of the same syllable revealed that shFoxP2-injected pupils sang syllables slightly, but significantly, more variably than control pupils or tutors (Figure 4C). As expected, shControl-injected pupils, shGFP-injected pupils, and tutors performed their syllables with equal stability (Figure 4C).

pbio-0050321-g004: Variability of Syllable Production in FoxP2 Knockdown PupilsEach vertical column shows the sonograms of five different renditions of the same syllable (scale bar indicates 100 ms, frequency range = 0–8,600 Hz). Each syllable, labeled by a roman numeral, was selected from a different bird. Of note, the first three syllables in (A) (syllables I, II, and III) were imitated from the same tutor as the corresponding syllables in (B) (syllables I′, II′, and III′).(A) FoxP2 knockdown pupils. Two vertical lines mark the beginning and the end of the longest rendition of each syllable to visualize variability of syllable duration (particularly evident in syllables II and IV). Also note the variability in acoustic structure between different renditions of the same syllable (e.g., FM of syllable I, shape and frequency of first element of syllable III, and PG of last element of syllable IV).(B) Syllable duration is relatively invariant in control pupils, as indicated by the vertical lines marking the beginning and the end of each syllable. Acoustic structure is also stable across syllable renditions.(C) Acoustic variability of syllables from rendition to rendition was higher in shFoxP2-injected than in control pupils (shGFP and shControl injections), as indicated by significantly lower syllable identify scores (two-tailed Mann-Whitney U test, *p < 0.05; Bonferroni-corrected α-level). Control and tutor birds sang with comparable variability (two-tailed Mann-Whitney U test, p > 0.8; tutors n = 6, on average 5 syllables per animal; shControl n = 7 animals, on average 4 syllables per animal; shGFP n = 3, on average 5 syllables per animal).(D) Syllable duration varied more from rendition to rendition in knockdown pupils (shFoxP2) than in controls (shControl and shGFP) and tutors, as indicated by a higher mean coefficient of variation of syllable duration (±SEM, two-tailed Mann-Whitney U test, **p < 0.001; Bonferroni-corrected α-level; no difference between tutors, shControl-injected, and shGFP-injected animals, p > 0.7, same animals as [C]).

Mentions:
The comparison of sonograms from different renditions of the same syllable revealed that knockdown pupils sang their syllables in a more variable fashion than control pupils (Figure 4A and 4B). Both the spectral (syllables I and III) and the temporal domain (syllables II and IV) were affected. Of note, the first three syllable examples shown in Figure 4A and 4B (syllables I, II, and III and I′, II′, and III′), stem from different animals, but were learned from the same tutor. To quantify the acoustic variability of syllables, we used the syllable identity score mentioned above. Pairwise comparison between different renditions of the same syllable revealed that shFoxP2-injected pupils sang syllables slightly, but significantly, more variably than control pupils or tutors (Figure 4C). As expected, shControl-injected pupils, shGFP-injected pupils, and tutors performed their syllables with equal stability (Figure 4C).

Bottom Line:
Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development.Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song.Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.

ABSTRACTThe gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.